Method for stabilization and delivery of therapeutic molecules

ABSTRACT

A method of treatment is disclosed, comprising administering a composition of Cyclodextrin and reduced, nanonized L-Glutathione to a patient in need of treatment, wherein the L-Glutathione molecule is non-acetylated, non-Esterified, and non-fatty acid attached.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/240,871, filed Aug. 18, 2016, which is a continuation-in-part of U.S.patent application Ser. No. 14/275,625, filed May 12, 2014, which is adivisional of U.S. patent application Ser. No. 13/526,332, filed Jun.18, 2012, which claims priority to U.S. Provisional Patent ApplicationNo. 61/497,869 filed on Jun. 16, 2011 all of which are incorporatedherein by reference in their entireties.

FIELD

The subject matter disclosed herein relates to molecular stabilizationand delivery using Cyclodextrin and more particularly relates tostabilization and delivery of Glutathione and other therapeutic orbio-enhancing molecules.

BACKGROUND

Glutathione (c-glutamylcysteinylglycine, GSH) is the major thiolatedsmall peptide present in living cells. Due to its reducing andnucleophilic properties, GSH acts as a redox buffer, thus preventingoxidative damage. Glutathione depletion has been observed in a number ofdisease conditions including lung and neurological diseases such asacute respiratory-disease, and Parkinson's-disease, respectively.Glutathione is indicated in the treatment of alcohol and drug poisoning,as well as for protection against toxicity induced by cytotoxicchemotherapy and radiation trauma and also in the treatment ofAIDS-associated cachexia. However, due to the chemical and enzymaticdegradation of the peptide in the jejunum, Glutathione is typicallyadministered intravenously. Additionally, the thiol group of thecysteine moiety in Glutathione is susceptible to enzymatic(c-glutamyl-transpeptidase) and non-enzymatic pH-dependent oxidation,leading to rapid degradation into non-active products. Therefore, thedevelopment of a technological approach that non-toxically stabilizesthe Glutathione molecule against oxidation and bypasses the digestivesystem would increase the use and clinical value of Glutathione. Thistechnology would also enhance the use of other therapeutic andbio-enhancing molecules.

SUMMARY

From the foregoing discussion, it should be apparent that a need existsfor a composition and method that non-toxically stabilizes a therapeuticor bio-enhancing molecule including but not limited to Glutatione.Beneficially, such a composition and method would provide transdermal,transmucosal, or other non-digestive and non-intravenous delivery.Accordingly, the composition and method provided herein have beendeveloped to provide for stabilization and delivery of Glutathione andother natural molecules.

Provided herein is a treatment method comprising administering to apatient in need of treatment a composition comprising gammaCyclodextrin, reduced, nanonized L-Glutathione, and an antioxidant,wherein the reduced L-Glutathione is non-acetylated, non-Esterified, andnon-fatty acid attached.

In certain embodiments the Cyclodextrin is gamma Cyclodextrin. Thenatural molecule sometimes comprises one or more of L-Glutathione andnanonized reduced Glutathione (RealGSH™). In some embodiments thenatural molecule comprises at least one of a protein, a fragmentthereof, and a polypeptide.

In some embodiments the natural molecule comprises at least one ofnucleic acid and fragment thereof. The fragment thereof may comprise atleast one of oligonucleotide, DNA, and RNA.

In certain embodiments the antioxidant comprises at least one ofalpha-Lipoic Acid, Ascorbic acid, Uric acid, beta-Carotene,alpha-Tocopherol, dimethylethanolamine (DMAE), CoEnzyme Q10, vitamin E,Carnosine, colloidal silver, and the enzymes catalase, superoxidedismutase, and peroxidase. The carrier sometimes comprises one or moreof a liquid, a spray, an aerosol, a cream, a tablet, a capsule, asuppository, a lotion, an aqueous solution, a powder, a paste, anointment, a jelly, a wax, an oil, a lipid, a lipid (cationic or anionic)containing vesicle (such as Lipofectin™), a DNA conjugate, an anhydrousabsorption paste, an oil-in-water and water-in-oil emulsion, an emulsiona carbowax (polyethylene glycols of various molecular weights), asemi-solid gel, and a semi-solid mixture containing carbowax.

In some embodiments of the method herein provided the composition isformulated for sustained delivery. The composition is sometimes appliedto one or more of the skin, mucosa, nose, eye, and lung.

Further provided herein is a method of treatment comprising, in someembodiments, administering a composition of gamma Cyclodextrin,nanonized, reduced Glutathione that is non-acetylated, non-Esterified,and non-fatty acid attached, and an antioxidant to a patient to treat aspecific condition. The patient may be an animal selected from the groupconsisting of mammal, bird, reptile, amphibian, and fish. The mammal issometimes a human.

In certain embodiments the condition to be treated comprises one or moreof alcohol or drug poisoning, intoxication, alcohol “hang over,”toxicity induced by cytotoxic chemotherapy, radiation trauma,AIDS-associated cachexia, HIV Aids, shingles, frostbite, heavy metalpoisoning, burns including laser burn, sun burn, traumatic burn, thermalburn, chemical burn, acne, pressure sore, autism, scar tissue,Parkinson's disease, hepatitis B, hepatitis C, upper respiratory virusinfections (cold), cystic fibrosis, insect bites (mosquito, spider,etc.), pain in limbs, neuropathy, Reflex Sympathetic Dystrophy (RSD),rheumatoid arthritis, multiple sclerosis, osteoarthritis, psoriasis,psoriatic arthritis, jet lag, kidney disease (CRF, CKD), akathisia, andtardive dyskinesia.

In various embodiments the condition to be treated comprises one or moreof obesity, decreased immunity, inflammation, angina, heart disease, andcardiac reperfusion injury, lung-and-neurological-diseases such as acuterespiratory-disease, emphysema, pulmonary fibrosis and associated musclewasting, asthma, migraine headaches; Parkinson's-disease, herpes zoster,HSV, hepatitis B&C, and influenza, fibromyalgia;osteoporosis/osteomalacia, cancer including but not limited to brain,head and neck, thyroid, lung, esophagus, stomach, intestine, liver,pancreas, kidney uterine, ovarian, prostate, leukemia (acute andchronic), lymphoma, multiple myeloma, and others, systemic sclerosis(scleroderma) syndrome, sepsis, trauma, wrinkles, sagging skin, acne,atopic dermatitis and eczema, athletic overtraining and muscle fatigue;schizophrenia, bipolar disorder, major depressive disorder, dementia,autism, Attention Deficit Hyperactive Disorder (ADHD); overdose ofacetaminophen, low energy, drug toxicity, eye problems includingcataracts, glaucoma, macular degeneration, macular dystrophy, diabeticretinopathy, decreased visual acuity, diabetic retinopathy, and contrastsensitivity; biomolecule imbalances resulting from traumatic head injuryor other causes, and infertility in men and women.

Also provided herein is a method for stabilizing reduced L-Glutathioncomprising bringing reduced L-Glutathione and an antioxidant in contactwith solubilized Cyclodextrin in a polar solution to form a complex ofreduced L-Glutathione and antioxidant compound and Cyclodex, adjustingthe pH to a range compatible with preserving the complex, and exposingthe complex to Ultrasonic waves sufficient to create complexnanoparticles of sizes in the range of from 2 nanometers to 200nanometers. The Cyclodextrin is sometimes gamma-Cyclodextrin. In someembodiments the polar solution is aqueous. The polar solution of areaction mixture may comprise ascorbic acid and Benzalkonium chloride,and the reaction mixture may be capped under vacuum after mixing at a pHin the range of 3.0 to 7.0. The antioxidant is sometimes a solublecompound having antioxidant activity and comprising a mixture of two ormore of ascorbic acid, ascorbic acid derivatives, L-Cysteine, N-AcetylCysteine, L-Carnitine, Acetyl-L-carnitine, Riboflavine and Curcuminoids.The antioxidant may be in the range of 0.001 mole and 100 moles per moleof reduced L-Glutathione and in certain embodiments the antioxidant isnot less than 0.01 mole and not more than 10 moles per mole of reducedL-Glutathione.

Further provided herein is an embodiment of a kit for topicalapplication, the kit comprising a complex of gamma Cyclodextrin andreduced, nanonized, L-Glutathione, which is non acetylated,non-Esterified, and non-fatty acid attached, a container for dispensingthe composition, a composition applicator and one or more ofinstructions for use.

Reference throughout this specification to “some embodiments,” “certainembodiments,” “various embodiments” or similar language means that aparticular feature, structure, or characteristic described in connectionwith the embodiment is included in at least one embodiment. Thus,appearances of the phrases “in some embodiments,” “in certainembodiments,” “in various embodiments,” and similar language throughoutthis specification may, but do not necessarily, all refer to the sameembodiment, but mean “one or more but not all embodiments” unlessexpressly specified otherwise. The terms “including,” “comprising,”“having,” and variations thereof mean “including but not limited to”unless expressly specified otherwise. An enumerated listing of itemsdoes not imply that any or all of the items are mutually exclusiveand/or mutually inclusive, unless expressly specified otherwise. Theterms “a,” “an,” and “the” also refer to “one or more” unless expresslyspecified otherwise.

Furthermore, the described features, structures, or characteristics ofthe embodiments may be combined in any suitable manner. One skilled inthe relevant art will recognize, however, that embodiments may bepracticed without one or more of the specific details, or with othermethods, components, materials, and so forth. Rather, language referringto the features and advantages is understood to mean that a specificfeature, advantage, or characteristic is included in at least oneembodiment. Thus, discussion of the features and advantages, and similarlanguage, throughout this specification may, but does not necessarily,refer to the same embodiment. In other instances, well-known protocols,reagents, materials, or operations are not shown or described in detailto avoid obscuring aspects of an embodiment.

These features and advantages of the embodiments will become more fullyapparent from the following description and appended claims, or may belearned by the practice of embodiments as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. FIG. 1B is a graph depicting venous blood levels of GSH of amale 65 years old after administering 200 mg of gamma-Cyclodextrin/GSHcomplex on forearm. Samples were drawn as indicated at time in FIG. 1A.

FIG. 2A-2B. FIG. 2B is a graph depicting venous blood level of GSH of afemale about 28 years old after administering 200 mg ofgamma-Cyclodextrin/GSH complex on forearm. Samples were drawn asindicated at time in FIG. 2A.

FIG. 3 depicts the results of an independent laboratory analysis of theraw topical stabilized GSH, without preservatives, as provided herein.The gel is the result of stabilizing the highly reactive cysteine moietyof GSH in a gamma-Cyclodextrin ring using high energy waves in an oxygenenvironment. The samples were sent via U.S. Mail in test tubes with fullexposure to atmospheric oxygen.

FIG. 4 depicts the toroid structure of Cyclodextrin.

FIG. 5A/B depicts the study design for evaluation of the safety andefficacy of topical Glutathione stabilized in a gamma-Cyclodextrin ringstructure (Example 11).

FIG. 6A-6B. FIG. 6A is a graph depicting a dose-based comparison ofglutathione absorption at doses of 50 mg/ml, 100 mg/ml and 200 mg/ml atincremental times from pre-dose to 180 minutes post-dose as indicated inFIG. 6B (Example 9).

FIG. 7A-7B. FIG. 7A is a graph depicting a dose-based comparison ofblood levels of superoxide dismutase at doses of 50 mg/ml, 100 mg/ml,and 200 mg/ml at incremental times from pre-dose to 180 minutespost-dose as indicated in FIG. 7B (Example 9).

FIG. 8A-8B. FIG. 8A is a graph depicting a dose-based comparison ofblood levels of glutathione peroxidase at doses of 50 mg/ml, 100 mg/ml,and 200 mg/mg at incremental times from pre-dose to 180 minutespost-dose as indicated in FIG. 8B (Example 9).

FIG. 9A-9B. FIG. 9A is a graph depicting a dose-based comparison ofblood levels of lipid peroxidase at doses of 50 mg/ml, 100 mg/ml, and200 mg/mg at incremental times from pre-dose to 180 minutes post-dose asindicated in FIG. 9B (Example 9).

FIG. 10 is a series of bar graphs depicting urinary output andcalculated urinary output of lead, mercury, and arsenic in 24 treatedvolunteers, within a 34 volunteer study, before and after treatment withGSH, as measured by Electrothermal (Flameless) AAS and Mercury Hydrideprocedure through Mountain Star Clinical Laboratories. (Example 10).

FIG. 11 is a bar graph depicting a numerical summary of urinary outputand calculated urinary output of lead, mercury, and arsenic in 24treated volunteers, within a 34 volunteer study, before and aftertreatment with GSH, as measured by Electrothermal (Flameless) AAS andMercury Hydride procedure through Mountain Star Clinical Laboratories.(Example 10).

FIG. 12 is a series of bar graphs depicting urinary output andcalculated urinary output of lead, mercury, and arsenic in a 10volunteer placebo group, within a 34 volunteer study, beforeintervention, after receiving a placebo, and after treatment with GSH,as measured by Electrothermal (Flameless) AAS and Mercury Hydrideprocedure through Mountain Star Clinical Laboratories. (Example 10).

FIG. 13 is a bar graph depicting a numerical summary of urinary outputand calculated urinary output of lead, mercury, and arsenic in a 10volunteer placebo group before intervention, after receiving a placeboand after treatment with GSH, as measured by Electrothermal (Flameless)AAS and Mercury Hydride procedure through Mountain Star ClinicalLaboratories. (Example 10).

FIG. 14 is a reproduction of a formula worksheet for the production of astabilized glutathione-Cyclodextrin complex (Example 1).

DETAILED DESCRIPTION

Herein provided is a composition and method of treatment for protectingand stabilizing therapeutic and bio-enhancing molecules, includingGlutathione, by means of a gamma-Cyclodextrin complex. The compositioncomprising this complex may optionally include other molecules, fornon-limiting example, antioxidants such as Alpha-Lipoic Acid, Ascorbicacid, Uric acid, Betacarotens, alphaTocopherol, DMAE and CoEnzyme Q10.The gamma-Cyclodextrin additionally facilitates transdermal andtransmucosal delivery of the molecules, thus bypassing the digestivetract and eliminating the need for intravenous administration.Transdermal stabilized glutathione is a novel form of Glutathione thathas been stabilized using encapsulation in gamma-Cyclodextrin ringstructures that prevents oxidation of the reduced Glutathione byatmospheric oxygen.

Antioxidants

An antioxidant is a molecule capable of inhibiting the oxidation ofother molecules. Oxidation is a chemical reaction that transferselectrons from a substance to an oxidizing agent. Oxidation reactionscan produce free radicals. In turn, these radicals can start chainreactions. When the chain reaction occurs in a cell, it can cause damageor death. Antioxidants terminate these chain reactions by removing freeradical intermediates, and inhibit other oxidation reactions. They dothis by being oxidized themselves, so antioxidants are often reducingagents including thiols such as GSH, ascorbic acid, or polyphenols.Thiol groups exist at a concentration of approximately 5 mM in animalcells. Glutathione reduces disulfide bonds formed within cytoplasmicproteins to cysteines by serving as an electron donor. In the process,Glutathione is converted to its oxidized form Glutathione disulfide(GSSG).

Although oxidation reactions are crucial for life, they can also bedamaging and oxidative stress appears to be an important part of manyhuman diseases. Therefore, plants and animals maintain complex systemsof multiple types of antioxidants, such as GSH, vitamin C, and vitamin Eas well as enzymes such as catalase, superoxide dismutase, and variousperoxidases. Low levels of antioxidants, or inhibition of theantioxidant enzymes, cause oxidative stress and may damage or killcells.

Cyclodextrin

Cyclodextrins (sometimes called cycloamyloses) are a family of compoundsmade up of sugar molecules bound together in a ring (cyclicoligosaccharides) and are produced from starch by means of enzymaticconversion. Cyclodextrins are used in food, pharmaceutical, and chemicalindustries, as well as agriculture and environmental engineering.

Cyclodextrins are composed of 5 or more (1,4)-linked α-D-glucopyranoseunits. Topologically, Cyclodextrins form a torus with a hydrophobicinterior and a hydrophilic exterior. Typical Cyclodextrins contain anumber of glucose monomers ranging from six to eight units in a ring,creating a cone shape. Alpha-Cyclodextrin is a six membered sugar ringmolecule, beta-Cyclodextrin is a seven sugar ring molecule, andgamma-Cyclodextrin in an eight sugar ring molecule. Cyclodextrins can betopologically represented as toroids with the larger and the smalleropenings of the toroid exposing to the solvent secondary and primaryhydroxyl groups respectively. (See FIG. 4.) Because of this arrangement,the interior of the toroid is not hydrophobic, but considerably lesshydrophilic than the aqueous environment and thus able to host otherhydrophobic molecules. In contrast, the exterior is sufficientlyhydrophilic to impart water solubility to Cyclodextrins (or theircomplexes).

This allows Cyclodextrins to act as host molecules that form inclusioncomplexes with hydrophobic guest molecules. Cyclodextrins are known toinfluence the percutaneous absorption of therapeutic agents by both asolubilizing action on the drug thus increasing its availability at theabsorption site and by an interaction with the free lipids present inthe stratum corneum resulting in improvement of transdermal penetrationof therapeutic agents.

The formation of the inclusion compounds greatly modifies the physicaland chemical properties of the guest molecule, mostly in terms of watersolubility. Thus, inclusion compounds of Cyclodextrins with hydrophobicmolecules are able to penetrate body tissues, and can be used to releasebiologically active compounds under specific conditions. The mechanismof controlled degradation of such complexes is sometimes based on pHchange of solutions, leading to the cleavage of hydrogen or ionic bondsbetween the host and the guest molecules. Alternative means for thedisruption of the complexes may involve heating or the action of enzymesable to cleave α-1,4 linkages between glucose monomers.

In Cyclodextrin inclusion one or more guest molecule interacts with thecavity of a Cyclodextrin molecule to form a stable association.Molecules or functional groups of molecules that are less hydrophilicthan water can be included in the Cyclodextrin cavity in the presence ofwater. The “guest molecules” may fit, at least partly, into theCyclodextrin cavity. The cavity sizes as well as possible chemicalmodifications determine the affinity of Cyclodextrins to the variousmolecules. In the case of some low molecular weight molecules, more thanone guest molecule may fit into the cavity. Conversely, some highmolecular weight molecules may bind more than one Cyclodextrin molecule.Therefore a 1:1 molar ratio is not always achieved. Gamma-Cyclodextrin,as provided herein, exhibits compatibility with Glutathione, andprotects the Glutathione molecule sufficiently to usefully extend theGlutathione half-life in the bloodstream.

In the solid state the guest molecule is molecularly dispersed in theCyclodextrin matrix, even with gaseous guest molecules. Thus, the guestmolecule is effectively protected against any type of reaction, exceptwith Cyclodextrin's hydroxyls. In aqueous solution the concentration ofa poorly soluble guest molecule in the dissolved phase increasessignificantly. Reactivity of the guest molecule decreases in most cases.

Cyclodextrins are able to form inclusion complexes with a broad range ofhydrophobic molecules, with the larger gamma-Cyclodextrin accepting morebulky compounds. As provided herein, Cyclodextrins andgamma-Cyclodextrin in particular can form compounds with peptides,polypeptides, proteins, amino acids, nucleic acids, polynucleotides,DNA, and RNA Anti-oxidants such as ascorbic acid, carnosine,alpha-Lipoic Acid, DMAE, CoEnzuyme Q10 and other molecules such ascolloidal silver may enhance the protective function of the complex andperform various other functions.

Cyclodextrin complexation of a drug may increase drug stability,sustaining the release and minimizing the photodegradation of acomplexed drug. Cyclodextrin complexation has utility in improving thechemical, physical and thermal stability of drugs. Chemical reactionsare necessary in order for an active molecule to degrade upon exposureto oxygen, water, radiation or heat. When a molecule is entrapped withinthe Cyclodextrin cavity, it is difficult for the reactants to diffuseinto the cavity and react with the protected guest.

The stabilized Cyclodextrin-Glutthione compound comes in the form of agel that is applied topically. The Cyclodextrin ring structures arebroken down by naturally occurring enzymes on the skin and the reducedglutathione is absorbed transdermally and enters the bloodstream.

Glutathione

Glutathione (GSH) as provided herein is natural, non-esterified,non-acetylated, and non-fatty acid attached, fostering highbioavailability. Gluthatione is a tripeptide that contains an unusualpeptide linkage between the amine group of cysteine and the carboxylgroup of the glutamate side-chain. It is an antioxidant, preventingdamage to various cellular components caused by reactive oxygen speciessuch as free radicals and peroxides. Glutathione is the most abundantlow molecular weight thioltripeptide synthesized in cells and helps tomaintain other antioxidants (such as Vitamin C) in the active reducedform. Clinical use of Glutathione in medicine has been limited becauseof its unstable nature due to the cysteine moiety of the Glutathione.Thus, if Glutathione is given intravenously, much of the Glutathione isoxidized into GSSG in the IV-bag during storage, transport or whilebeing infused. Nebulized forms have also been used but the smell andtaste result in poor patient compliance and it is counterintuitive tosuggest that aerosolizing a compound that is highly reactive in thepresence of atmospheric oxygen is an effective strategy.

In vivo, Glutathione is found almost exclusively in its reduced form,since the enzyme that reverts it from its oxidized form, Glutathionereductase, is constitutively active and inducible upon oxidative stress.In fact, the ratio of reduced Gluathione to oxidized Glutathione withincells is often used as a measure of cellular toxicity. Glutathione helpsprevent damage to cells by neutralizing harmful molecules generatedduring energy production. Glutathione also plays a role in processingmedications and cancer-causing compounds (carcinogens), and buildingDNA, proteins, and other important cellular components.

Glutathione is known as a substrate in both conjugation reactions andreduction reactions-catalyzed by glutathione S-transferase enzymes-incytosol, microsomes, and mitochondria. However, it is also capable ofparticipating m non-enzymatic conjugation with some chemicals.Glutathione participates in leukotriene synthesis and is a cofactor forthe enzyme glutathione peroxidase. It is also important as a hydrophilicmolecule that is added to lipophilic toxins and waste in the liverduring biotransformation before they can become part of the bile.Glutathione also assists in the detoxification of methylglyoxal, a toxinproduced as a by-product of metabolism.

Low Glutathione is strongly implicated in wasting and negative nitrogenbalance, as seen in cancer, AIDS, sepsis, trauma, burns and evenathletic overtraining. Glutathione supplementation can oppose thisprocess, and in AIDS, for example, result in improved survival rates.Schizophrenia and bipolar disorder are associated with loweredGlutathione. Accruing data suggest that oxidative stress may be a factorunderlying the pathophysiology of bipolar disorder (BD), majordepressive disorder (MDD), and schizophrenia (SCZ). Glutathione is themajor free radical scavenger in the brain. Diminished Glutathione levelselevate cellular vulnerability towards oxidative stress; characterizedby accumulating reactive oxygen species. Replenishment of Glutathioneusing N-acetyl cysteine has been shown to reduce symptoms of thesedisorders.

Glutathione is an antidote to overdose in the case ofN-acetyl-p-benzoquinone imine (NAPQI), the reactive cytochromeP450-reactive metabolite formed by paracetamol (known in the U.S. asacetaminophen), that becomes toxic when Glutathione is depleted by anoverdose of acetaminophen. Glutathione conjugates to NAPQI and helps todetoxify it. In this capacity, it protects cellular protein thiolgroups, which would otherwise become covalently modified; when allGlutathione has been spent, NAPQI begins to react with the cellularproteins, killing the cells in the process.

Preliminary results on isolated cells indicate Glutathione changes thelevel of reactive oxygen, which may reduce cancer development.Additional evidence indicates that adequate levels of Glutathione helpto control the level of Tumor necrosis factor (TNF)-a group of cytokinesthat can cause cell death. However, once a cancer has already developed,elevated levels of GSH in tumor cells confers resistance to a number ofchemotherapeutic drugs, and thus protects cancerous cells in bonemarrow, breast, colon, larynx, and lung cancers.

Excess glutamate at synapses, which may be released in conditions suchas traumatic brain injury, can prevent the uptake of cysteine, anecessary building-block of Glutathione. Without the protection fromoxidative injury afforded by Glutathione, cells may be damaged orkilled.

Raising Glutathione levels through direct supplementation of Glutathioneis difficult. Research suggests that Glutathione taken orally is brokendown by digestive enzymes and not well absorbed across thegastrointestinal tract. Additionally, natural GSH can be quite rapidlyoxidized upon exposure to air. Some attempts have been made to stabilizeGlutathione by acetylating or esterifying the Glutathione thiol group.

The composition provided herein obviates the necessity of acetylating,esterifying, or otherwise modifying the Glutathione. The disclosedcomposition comprises a complex of reduced L-Glutathione andgamma-Cyclodextrin, as discussed below, which protects the Glutathionefrom degradation and oxidation without the necessity of altering thenatural Glutathione molecule.

Nanonized Glutathione Cyclodextrin Complex

Nanonization of a pH stabilized Glutathione Cyclodextrin complex mayfacilitate rapid absorption into the bloodstream as discussed below. Insome embodiments sodium hydroxide, hydrochloric acid or other acid isadded to the stabilized Glutathione, antioxidant, and GammaCyclodextrine to adjust the pH of the solution. In various embodimentsthe pH is adjusted to a pH of not less than 4.00 and not more than 7.8.In certain embodiments the adjusted pH is not less than 5.00 and notmore than 7.2. The solution may be nanonized using Ultrasonic waves asknown in the art in the range of about 100 Watts. The Ultrasonic wavesare sometimes applied for not less than 1 minute and not more than 10minutes. In certain embodiments the Ultrasonic waves are applied for notless than 3 minutes and not more than 6 minutes.

The size of the resulting nanoparticles may be in the range of between 2nanometers and 200 nanometers. In certain embodiments the nanoparticleshave a size in the range of 2 to 20 nanometers, 20 to 40 nanometers, 40to 60 nanometers, 60 to 80 nanometers, 80 to 100 nanometers, 100 to 120nanometers, 120 to 140 nanometers, 140 to 160 nanometers, 160 to 180nanometers, and 180 to 200 nanometers. In some embodiments thenanoparticles may be of mixed sizes.

Therapeutic Administration and Formulations

The composition provided herein may be formulated as a liquid, cream,solid, lotion, oil, emulsion, spray, aerosol, dissolving strip, bolus,suppository, tablet, capsule, or other formulation using compounding andother methods known in the art. In certain embodiments a guest moleculesuch as gamma-Cyclodextrin and GSH are combined in an aqueous solutioncomprising ascorbic acid and Benzalkonium chloride, and capped undervacuum after mixing at a pH of between 3.0 and 7.0. In some embodimentsthe pH is 5.5. In certain embodiments the pH is from 3.0 to 4.0, from4.0 to 5.0, from 5.0 to 6.0, or from 6.0 to 7.0.

The percentage of Cyclodextrin may be from 1% to 27%. The percentage ofCyclodextrin may sometimes be from 1% to 5%, from 5% to 10%, from 10% to15%, from 15% to 20%, from 20% to 25% and from 25% to 30%. Thepercentage of Glutathione or other guest molecule may be from 0.1% to80%. In certain embodiments the percentage of Glutathione or other guestmolecule is from 0.1% to 1%, from 1% to 5%, from 5% to 10%, from 10% to15%, from 15% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%,from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to 85%, from85% to 90%, and from 90 to 95%.

In some embodiments Cyclodextrin is 0.116 M. In various embodimentsGlutathione is 3.1 M. The molar ratio of Glutathione to Cyclodextrin issometimes 26 to 1. The molar ratio of GSH to Cyclodextrin may be between1 to 15 or less and 30 to 1 or more. By way of non-limiting example, themolar ratio of Glutathione or other guest molecule to Cyclodextrin maybe: 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1 to 1, or anyfraction of the foregoing ratios. In certain embodiments the molar ratioof GSH or other guest molecule to Cyclodextrin is 1.1 to 1, 1.2 to 1,1.3 to 1, 1.4 to 1, 1.5 to 1, 1.6 to 1, 1.7 to 1, 1.8 to 1, 1.9 to 1, 2to 1, 3 to 1, 4 to 1, 5 to 1, 6 to 1, 7 to 1, 8 to 1, 9 to 1, 10 to 1,11 to 1, 12 to 1, 13 to 1, 14, to, 15 to 1, 16 to 1, 17 to 1, 18 to 1,19 to 1, 20 to 1, 21 to 1, 22 to 1, 23 to 1, 24 to 1, 25 to 1, 26 to 1,27 to 1, 28 to 1, 29 to 1, and 30 to 1, or any fraction of the foregoingratios. In certain embodiments the concentration of Glutathione is 950mg/ml. In various embodiments the concentration of Cyclodextrin 150mg/ml. The Cyclodextrin as provided above is sometimesgamma-Cyclodextrin. In some embodiments the combined concentrationpercentage is 95% GSH in 15% gamma-Cyclodextrin.

Various natural molecules may be combined with Cyclodextrin m a similarfashion to form inclusion complexes. In certain embodiments antioxidantsand other molecules may be added, including but not limited to Ascorbicacid, Alpha-Lipoic Acid, Uric acid, alpha Tocopherols, beta Carotenes orany other antioxidant molecules.

Specifically the composition of this invention which contains a reducedL-Glutathione in a stabilizing solution which may contain anantioxidant. The antioxidant may be any soluble compound havingantioxidant activity which may be mixtures of two or more of ascorbicacid, ascorbic acid derivatives, L-Cysteine, N-Acetyl Cysteine,L-Carnitine, Acetyl-L-carnitine, Riboflavine and Curcuminoids. In someembodiments the antioxidant is not less than 0.001 mole and not morethan 100 moles per mole of reduced L-Glutathione, and may be not lessthan 0.01 mole and not more than 10 moles of reduced L-Glutathione.

A method for solubilizing and stabilizing is described. The methodcomprising bringing reduced L-Glutathione with another compound ofantioxidant on contact with solubilized gamma-Cyclodextrin in a polarsolution preferably aqueous. After such mixing a complex of reducedL-Glutathione and antioxidant compound and gamma-Cyclodextrin is formed.

The composition and method provided herein may be employed to treat avariety of conditions including without limitation: alcohol or drugpoisoning, intoxication, alcohol “hang over,” toxicity induced bycytotoxic chemotherapy, radiation trauma, AIDS-associated cachexia, HIVAids, shingles, frostbite, heavy metal poisoning, burns including laserburn, sun burn, traumatic burn, thermal burn, chemical burn, acne,pressure sore, autism, scar tissue, Parkinson's disease, hepatitis B,hepatitis C, upper respiratory virus infections (cold), cystic fibrosis,acne, insect bites (mosquito, spider, etc), pain in limbs, neuropathy,Reflex Sympathetic Dystrophy (RSD), rheumatoid arthritis, multiplesclerosis, osteoarthritis, psoriasis, psoriatic arthritis, jet lag,kidney disease (CRF, CKD), and akathisia, tardive dyskinesia, obesity,decreased immunity, inflammation, angina, heart disease, and cardiacreperfusion injury, lung- and neurological-diseases such as acuterespiratory-disease, emphysema, pulmonary fibrosis and associated musclewasting, asthma; cystic fibrosis, migraine headaches; Parkinson'sdisease, herpes zoster, HSV, hepatitis B & C, influenza, fibromyalgia,osteoporosis/osteomalacia, systemic sclerosis (scleroderma) syndrome,sepsis, trauma, wrinkles, sagging skin, acne, atopic dermatitis andeczema, athletic overtraining and muscle fatigue; schizophrenia, bipolardisorder, major depressive disorder, dementia, autism, Attention DeficitHyperactive Disorder (ADHD); overdose of acetaminophen, low energy, drugtoxicity, eye problems including cataracts, glaucoma, maculardegeneration, macular dystrophy, diabetic retinopathy, decreased visualacuity, diabetic retinopathy, and contrast sensitivity; biomoleculeimbalances resulting from traumatic head injury or other causes, andinfertility in men and women. The composition and method herein providedmay be used to treat cancer including but not limited to brain, head andneck, thyroid, lung, esophagus, stomach, intestine, liver, pancreas,kidney uterine, ovarian, prostate, leukemia (acute and chronic),lymphoma, multiple myeloma, and others.

The composition and method may be administered under physicianprescription or over the counter depending upon the natural molecule andother ingredients comprising the composition and upon the condition tobe treated. The route of administration is in accord with known methodsincluding without limitation; oral, sublingual, transdermal, cutaneous,subcutaneous, mucosal, transmucosal, inhalation, intralesional, buccal,or by sustained release systems as noted below. In some embodiments thecomposition as herein provided is administered via a small strip orother form of material that may dissolve in the mouth of the patient.This allows the convenience of a solid form therapy while retaining theadvantages of a sublingual or mucosal delivery. The enzymes of the humanmouth are capable of dissolving carbohydrates but not of breaking downpeptides or proteins or many types of organic molecule. Therefore thecomposition may be delivered directly to the bloodstream without beingexposed to digestive enzymes or crossing the intestinal barrier, andwithout the necessity for intravenous delivery.

An effective amount of composition to be employed therapeutically willdepend, for example, upon the specific composition, therapeuticobjectives, the route of administration, and the weight and condition ofthe patient. Accordingly, the therapist may titer the dosage and modifythe route of administration as required to obtain the optimaltherapeutic effect. The clinician may administer the composition until adosage is reached that achieves the desired effect. The progress of thistherapy may be monitored by conventional assays or by the assaysdescribed herein.

The therapeutic composition can be administered through the skin,mucosa, nose, eye, or lung, in formulations including a liquid, cream,lotion, oil, emulsion, gel, paste, powder, liquid or powder aerosol(lyophilized). The composition may be administered parenterally orsubcutaneously as desired. The composition may be administeredsystemically, and may be sterile, pyrogen-free and in a parenterallyacceptable solution having due regard for pH, isotonicity, andstability. These conditions are known to those skilled in the art.Briefly, dosage formulations of the compounds described herein areprepared for storage or administration by mixing the compound having thedesired degree of purity with physiologically acceptable carriers,excipients, or stabilizers, for example Cyclodextrin andgamma-Cyclodextrin. Such materials are non-toxic to the recipients atthe dosages and concentrations employed, and may include buffers such asTRIS HCl, phosphate, citrate, acetate and other organic acid salts;antioxidants such as ascorbic acid, carnosme, alpha-Lipoic Acid;peptides such as polyarginine, proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone;amino acids such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;counterions such as sodium and/or nonionic surfactants such as TWEEN,PLURONICS or polyethyleneglycol.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thecomposition provided, which matrices are in the form of shaped articles,films or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate),copolymers of L-glutamic acid and gamma ethyl-Lglutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as poly-D-(−)-3-hydroxybutyric acid.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37 degree C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

The dosage of the composition herein for a given patient will bedetermined by the therapist or physician taking into consideration thenatural molecule comprising the composition and various factors known tomodify the action of drugs including severity and type of disease, bodyweight, sex, diet, time and route of administration, other medicationsand other relevant clinical factors. Therapeutically effective dosagesmay be determined by either in vitro or in vivo methods.

An effective amount of the composition herein to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, the therapist may titer the dosage and modify theroute of administration as required to obtain the optimal therapeuticeffect. A daily dosage might range from about 0.001 mg/kg to up to 100mg/kg or more, depending on the factors mentioned above. In someembodiments the dosage is 50, 100, or 200 mg of GSH administered as atopical gel. In some embodiments the dose is administered twice daily,once in the AM and once in the PM. The clinician may administer thetherapeutic composition as provided herein until a dosage is reachedthat achieves the desired effect. The progress of this therapy may bemonitored by conventional assays or as described herein.

It will be appreciated that administration of therapeutic entities inaccordance with the compositions and methods herein may be administeredwith suitable carriers, excipients, and other agents that areincorporated into formulations to provide improved transfer, delivery,tolerance, and the like. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as Lipofectin™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures may be appropriate in treatments andtherapies in accordance with the present composition, provided that theactive ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration and as known in the art.

The embodiments may be practiced in other specific forms. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

EXAMPLES Example 1: Preparation of Gamma Cyclodextrin & Glutathione

Final Concentrations:

Glutathione 0.65 M (200 mg/ml)

Ascorbic acid 0.23 M (40 mg/ml)

Gamma Cyclodextrin 0.189 M (245 mg/ml)

Benzalkonium chloride 0.02%

1. Preparation:

1.1. Purified water 0.5 L was degassed by mixing in a capped 2 L filterErlenmeyer flak under vacuum for 30 min with a magnetic stirrer.

1.2. Vacuum was turned off, the flask was uncapped and 200 g ofL-Glutathione (Reduced form) was added into the flask. 40 g of Ascorbicacid was also added. Appropriate volume of Sodium hydroxide solution wasalso added to final ph oh about 5.5-6.5.

1.3. The flask was recapped, vacuum turned back on and the mixture wasmixed until dissolved to a clear solution.

1.4. 150 g of Gamma-cyclodextrin was added to flask after uncapped andvacuum turned off.

-   -   a. Purified water was added to about 1 L. The flask was        recapped, vacuum turned on and the mixture was mixed until        clear, about 1 h.    -   b. 0.4 ml of 50% Benzalkonium chloride was added and mixed well        also under vacuum.    -   c. Final solution was packed into air tight dispenser.        (See FIG. 14.)

Example 2: Blood Level of Glutathione

About 1 ml of 200 mg/ml GSH, 40 mg/ml Ascorbic Acid, 150 mg/ml GammaCyclodextrinin 0.02% Benzalkonium chloride was applied on the skin ofthe subject's forarm skin at time 0. A sample of 5 ml was drawn frommedian cubital vein of the same forearm at 15, 30, 45, and 60 min (FIG.2A/B). Samples were kept on ice and were shipped overnight to the labfor analysis of venous levels of reduced GSH.

Example 3: Glutathione Stability

Laboratory results on raw Glutathione stabilized in the disclosedgamma-Cyclodextrin ring structure showed that the Glutathione remained91% reduced after multiple transfers of location (FIG. 3).

Example 4: Clinical Observations on the New Topical Glutathione

Researcher self-administered topical Glutathione after receiving a lowblood Glutathione reading (FIG. 6A/B). Researcher observed an almostimmediate improvement in symptoms of fatigue and drowsiness. After 70+days a repeated intracellular Glutathione reading showed an increase tonormal (FIG. 7A/B). Clinically, researcher had lost about 10 pounds, andexperienced a noticeable increase in energy, along with a significantincrease in mental clarity.

Gel form topical Glutathione was then administered to patients enrolledin various mini-clinical trials. Clinically results included resolutionof light skin wrinkles at the application site, increase in energylevels and focus, dramatic increase in mental clarity, a 2.5 point (onaverage) drop in HbAlC with diabetic patients (in first 30days—approximately 10 patients were tested and showed a significantchange in HbAlC), a decrease in abnormal liver function tests inpatients with a large variety of liver problems, increase in stamina andexercise tolerance/endurance, an average 5 lb. weight loss in first 30days, self-reported clinical improvement of muscle and joint pain whenapplied to specific joints including hands, feet, and knees. Themini-clinical trial results were consistently observed by both thepatients themselves and the researcher.

TABLE 1 Results Patient Results Patent Results (% Control) (% Control)Reference Range Micronutrients Before After (greater than) AntioxidantsGlutathione 38 (Deficient) 50 >42% Cysteine 49 47 >41% Coenzyme Q-10 9286 (Deficient) >86% Selenium 84 79 >74% Vitamin E (A- 90 90 >84%tocopherol Alpha Lipoic Acid 90 89 >81% Vitamin C 62 63 >40% SPECTROX ™Total Antioxidant 73 >65% Function

Example 5: Reduced Glutathione (RealGSH™) Studied Benefits

Dosing Study—Though RealGSH™ can be made at concentrations permilliliter of 50 to 950 milligrams there seemed to be little differencein the higher doses versus 100 mg/ml. Dosing studies were performedusing the same person (prevented confounding via skin variations) andapplying several different doses. It was important to note that thisstudy continued to 4 hours but the levels did not level off at the endof the 4 hours—and may have continued to elevate.

Intracellular Levels Increase—with topical application for approximately5 weeks one 50 year old patient whose intracellular levels were low(tested through Spectracell™ out of Texas) showing signs of fatigue andmental fog, had a complete resolution of symptoms and then at 5 weeksshowed a significant rise in intracellular GSH levels with follow-uprepeat Spectracell™.

Burn Pain Reduction—Due to the molecular binding capability of the GSHmolecule researcher's noted a significant (usually 100%) almostinstantaneous decline in pain post burn (sunburn, household burn, orsunburn) when applied directly to the burn site. This is of courselimited to First and Second Degree burns. The burn also appears to heelmuch more quickly with less scarring.

Muscle and Joint Pain Reduction—On over 300 patients it was noted thatapproximately 99% had a dramatic decrease in overall pain with dailyuse, most within the first few minutes. Most were able to indicated theywished to stop all pain meds including narcotics and appeared to do sowithout any ill withdrawal effects or otherwise.

Detoxification Capabilities—36 patient double-blinded study. Startedwith 24 hour urine collected for Quantitative Heavy Metals, which wereall normal followed by a repeat 24 hour urine collected for QuantitativeHeavy Metals, which showed in more than 91% of cases that there was asignificant increase in heavy metal output in the urine when ourRealGSH™ was applied (we did not use a controlled amount or dose). The10 patient control showed no increase in their second urine but thenthey were crossed back over to the study group where they showed asignificant increase.

Alcohol Detoxification Capabilities—In repeat studies volunteers couldnot become intoxicated when RealGSH™ was applied before imbibing.Volunteers who got intoxicated (not applying the RealGSH™) who thenapplied the RealGSH™ would then become sober in approximately 34½minutes on average.

Improvement in Chronic Renal Failure—Two patients (one in her 70s andthe other in his early 30s) with diagnosed CRF (Chronic Renal Failurewith GFRs <30) both had improvement back to a normal GFR (>50) within amonth of daily use of RealGSH™

Improvement in Acne Pustule Formation, Reduction in Inflammation andScarring—In a 5 patient study with Grade 1-3 facial acne there wasdramatic decrease in pustule formation and inflammation plus noticeablefading of scars. This was documented with both questionnaires andclose-up digital photography.

Arthritis Symptom Improvement—Consistently patients noticed clinicalimprovement in their joint inflammation and increased range of motionupon application. This occurred with both weight bearing joints (kneesand ankles) and fine joints (such as in the hands). WE think this andmany of these other observations are due to the sticky nature of theRealGSH™ reduced glutathione at a molecular level apparently bindinginflammatory cytokines.

Increased Wellbeing—Vast majority of patients who used RealGSH™ reporteda marked improvement in their sense of well-being.

Increased Mental Clarity—Vast majority of patients who used RealGSH™reported a marked improvement in their mental clarity—this does not seemto be age related.

Depression Improvement—Vast majority of patients who used RealGSH™reported a marked improvement in their depression symptoms.

Reflex Sympathetic Dystrophy (RSD) Pain Resolution—In one patient (casereport pending) marked improvement in lower extremity Reflex SympatheticDystrophy (RSD) occurred with multiple daily applications of RealGSH™ tothe site. This is significant because RSD is considered to be one of themost painful disorders one can have and is basically considered almostuntreatable. This goes along with the other improvements in pain notedabove in #4. The pain reduction (from a 9+ to a 1-2) began on the firstday of therapy—was documented daily with questionnaires filled out bythe patient (a 53 year old teacher on her feet all day).

Rapid Improvement in HbAlC in Type 2 Diabetics—HbAlC is a 3-6 monthcellular “look back” on your average blood sugar levels. Withapplication of RealGSH™ in approximately 10 Type 2 diabetics reported asignificant (and somewhat unexplainable) drop in their HbAlC levels(from 0.7-2.0) would occur in the first 30-45 days. Despite RBC celllife questions (theoretically what we saw consistently should not havehappened in such a short time) we considered this to be a verysignificant benefit of RealGSH™, maybe one of the most important.

Marked Lowering of Triglyceride Levels (beyond the capabilities of astatin)—In 7 patients with hypertriglyceridemia formerly treated withvarious statins, after discontinuation of the statins and dailyapplication of the RealGSH™, an improvement of triglyceride levels (50points lower on average) were noted in the 7 patients. No side effectswere noted.

Improvement in LFTs in Hepatitis C patients in Liver Failure—In 2patients in liver failure (awaiting transplant) with LFTs (LiverFunction Tests) in the 10,000+ range, after 30 days of application ofRealGSH™, their LFTs returned to normal. No viral counts were obtainedpre or post application but studies continue.

Example 6: Second Degree Burn with Dramatic Healing after Application ofNanonized GSH; Case Report

A second degree burn affects the epidermis and the dermis, classified assuperficial or deep according to the depth of injury. The superficialtype involves the epidermis and the papillary dermis and ischaracterized by pain, edema, and the formation of blisters; it healswithout scarring. The deep type of burn extends into the reticulardermis, is pale and anesthetic, and results in scarring. Topical stableranoized GSH is a pharmaceutical grade product (RealGSH™ produced in theUSA by The Glute Group, LLC of Utah) that involves a Japanese(BioKyowa™) natural reduced glutathione (GSH) encased in a nano(γ-cyclodextrin ring from Cavamax™ in Germany) ring, the encasement ofwhich involves a process patent (currently pending). This has beenplaced in a sterile hydrous solution for topical application (from whichit may be easily applied). In testing this product caused no known sideeffect and its components (GSH and ring) are considered GRAS certifiedby the FDA.

A 50 year old woman accidentally received a second degree burn on herleft clavicle after lifting a pan of boiling water off her stove tooquickly. She rated her pain post burn as 10 (on a scale of 0-10) whenquestioned and so immediately applied a lavender essential oil toalleviate some of the pain but a few days later blisters had formed andthe pain was still an 8 especially with movement. Coincidentally shepresented to our office to take part in a detox trial being run on a newtopical stable nanonized GSH (RealGSH™) to determine heavy metalmovement in 24 hour urine samples with topical application. At oursuggestion she decided to apply it topically to the burn (numerousarticles on burns had shown how they could improve with GSH). Thefollowing are her diary notes of what happened afterwards:

“Day 1: Apr. 13, 2012

Pain level 8 (caused during movement through scar being pulled duringwalking, etc. She reported that she had to keep her shoulder still toavoid pain.).

First and second application: applied to the burn site every couple ofhours. By the second application the burn literally dried up and beganto flake off. At one point I lifted off a brownish flake that was thesize of a penny. I was shocked! Pain level had dropped to a 0-2 almostimmediately upon application.

Day 2: Apr. 14, 2012

Pain level 5 Caused during movement. Patient was able to begin moderatemovement of the shoulder. The blisters that had only begun to harden theday before had become small scabs. After the first application ofglutathione on the burn, one of the scabs fell off in researcher'sfingers. Images show that by the second application, the secondblister/scab began to lift off.

Day 3: Apr. 15, 2012

Pain level 0” The clinical picture showed a partial to full thicknessvery tender and painful second degree blistering burn with some escharformation when the patient first appeared. Clinical evaluation wasperformed daily thereafter with noticeable improvement but the mostsignificant finding is patient's sudden decrease in pain with theapplication of the RealGSH™. Of note the patient has a history offibromyalgia and adrenal insufficiency (all felt to be related topituitary dysfunction) but is only on natural therapies—her surgicalhistory is unremarkable. Patient's detox study results showedsignificant urinary arsenic output. Almost complete resolution (the burnturned to pink new skin on Day 3 as noted above and in photos) with nopain occurred by Day 3.

This case illustrates that the treatment not only helps reduce or evenalleviate pain from these burns but accelerates healing and reduces scarformation. Both glutathione and the cyclodextrin ring have been ruled asGRAS/E certified (Generally Regarded As Safe Effective) by the FDAGlutathione has been theorized in numerous review articles (includingNIH reviews) to perform a large number of healthy functions includingincrease photo-protection of sunburned cells but since there has notbeen a version that is actually stable and reduced and topical (now inconsideration the only way, other than intravenously, that reducedglutathione can be added to the body—when taken orally the oxidized orreduced version is immediately digested becoming almost useless or somepeople do not have the ability to make GSH) until RealGSH was developed.The size of the nano particles of RealGSH™ are 7.5-8.15 Angstroms indiameter which are readily and easily absorbed through the skin matrixwhere the cyclodextrin rings (actually just a sugar) are broken down byenzymes, allowing the GSH to readily move intracellularly or into theblood stream for a rapid response as seen in this patient.

Researcher recommends further study and clinical evaluations as thiscould become an important therapy in the treatment of first and seconddegree burns and possible prevention of partial burn advancement tothird degree especially in the area of post-burn pain relief.

Example 7: Reduction in HbAIC in Type 2 Diabetic with Topical StableNanonized Reduced Glutathione—A Case Report

Type 2 Diabetes is a condition characterized or caused by a decline infunctionality of the insulin receptors on the cell surface. GSH (reducedglutathione—the active form of glutathione) depletion has been shown toimpair glucose tolerance. The reverse then should hold true—increasingthe amount of GSH in the body should improve insulin receptor function.Topical stable nanonized GSH is a Patented pharmaceutical grade product(RealGSH™ produced in the USA by The Glute Group, LLC of Utah) thatinvolves a Japanese (BioKyowa™) natural reduced glutathione (GSH)encased in a nano (γ-cyclodextrin ring from Cavamax™ in Germany) theencasement of which involves a process patent (currently pending). Thisis then placed in a sterile hydrous solution for topical application(from which it is easily applied). This product causes no known sideeffect and its components (GSH and ring) are considered GRAS certifiedby the FDA Researchers believe from their previous work and otherresearch (NIH review articles, etc.), that reduced GSH has beendetermined to be significantly reduced in diabetics and has beenpostulated to lower glycosylated hemoglobin—but an easily utilizableeffective stable GSH has not been previously available.

A 64 year old male patient with Type 2 Diabetes (ten plus yearsduration) who was on no medications and was poorly controlled via dietwas given topical stable nanonized GSH at 200 mg/ml and advised to apply2 squirts twice a day (approximately I ml a day or 200 mg) for fifty(i.e. 54) days and then retested. No other medications or changesoccurred in his therapy during this time. His HbAIC dropped 0.7 point in30 days, a noticeable improvement.

Decreases of 2 points or greater have also been seen but are strictlyanecdotal at this point. If this were helpful in Type 2 Diabetes itwould be a relatively inexpensive, side effect free, highly beneficialtherapy that is easy to apply and helpful in many other ways (as ananti-oxidant, potential prevention against macular degeneration, etc.).

Example 8: Reflex Sympathetic Dystrophy Improved with Topical StableNanonized GSH—A Case Report

Reflex Sympathetic Dystrophy is one of the most painful and debilitatingcondition known in medicine. Complex Regional Pain Syndrome (CRPS), alsoknown as Reflex Sympathetic Dystrophy, is a chronic neurologicalsyndrome characterized by severe burning pain, pathological changes inbone and ski, excessive sweating, tissue swelling, and extremesensitivity to touch.

There are Two Types of CRPS—Type I and Type II. CRPS Type I (alsoreferred to as RSD)—involve cases in which the nerve injury cannot beimmediately identified. CRPS Type II (also referred to asCausalgia)—cases in which a distinct “major” nerve injury has occurred.CRPS is best described in terms of an injury to a nerve or soft tissue(e.g. broken bone) that does not follow the normal healing path. CRPSdevelopment does not appear to depend on the magnitude of the injury.The sympathetic nervous system seems to assume an abnormal functionafter an injury. Since there is no single laboratory test to diagnoseCRPS, the physician must assess and document both subjective complaints(medical history) and, if present, objective findings (physicalexamination). It is usually considered only treatable with painalleviation such as chromic narcotic use and other pain managementtherapies. Topical stable nanonized GSH is a patented pharmaceuticalgrade product (RealGSH™ produced in the USA by The Glute Group, LLC ofUtah) that involves a Japanese (BioKyowa™) natural reduced glutathione(GSH) encased in a nano (γ-cyclodextrin ring from Cavamax™ in Germany)ring, the encasement of which involves a process patent (currentlypending). This is then placed in a sterile hydrous solution for topicalapplication (from which it is easily applied). This product causes noknown side effect and its components (GSH and ring) are considered GRAScertified by the FDA Researchers believe from their own previous workand other research (NIH review articles, etc.) that reduced GSH hadaggressive molecular binding capabilities-enabling it to bind and removefrom the body (via the kidneys) just about anything that should not bethere (including, we hoped, inflammatory cytokines and pain mediators).GSH has also been determined to be reduced in neuropathic pain soelevating levels would hopefully alleviate the pain and RealGSH™ hasbeen shown upon application to immediately elevate skin and tissuelevels of GSH.

This patient was a 54 year old school teacher who had been a Type 2Diabetic for 20 years (oral controlled) and had received the diagnosisof RSD (CRPS Type I) ten years prior when she a underwent a second toeamputation on the right foot. Her blood sugars were well controlled buther pain was becoming intolerable and she had been considering a nerveblock and “spinal procedure” when she presented. The compounded topicalnanonized stable reduced GSH was prescribed with almost immediateimprovement (a daily questionnaire log was kept by the patient. Paindropped from a 9+ most days to 1-2 and became very tolerable. She had toapply the GSH every four hours but had no problem with application. Shereported being able to ambulate and stand on it (she taught math at ahigh school) and that it had changed her life. Her pain most days is now0-1 and she reports is very tolerable. This treatment shows promise asnew therapy to aid in the care of these patients.

Example 9: Response of Blood GSH Levels to Transdermal StabilizedGlutathione; Dosing Case Study

The purpose of this cohort was to measure Glutathione levels only, asmeasurements were for single doses and not extended treatment. Studiesfollowed the procedures of a complete and formal research protocol,available upon request from RealGSH™. One purpose was to determinewhether or not reduced glutathione penetrated the skin in a useableform.

The test subject was a 51 y/o cau male who had no previous exposure toGSH or GSH enhancing compounds applied topically, orally or otherwise.The test subject's past medical history was as follows:

Primary hypertension, primary hypercholesterolemia, cholelithiasis,gastric ulcers, kidney stones, diagnosed as “pre-diabetic”, tensionheadaches, migraine headaches, psoriasis, ankylosing spondylitis andintermittent arthritis. The subject had used Ibuprofen, and Extrastrength Tylenol used PRN for occasional headaches and was not usingvitamins or supplements. There was no report of past surgical history.Subject had no known allergies.

Baseline blood GSH levels were drawn as well as safety lab's consistingof CMP, urinalysis, CBC, lipid profile and HbAlC. 1 ml of the 50 mg/mlGSH gel was applied to an area of clean skin and a chronological seriesof blood samples were drawn to determine if plasma GSH levels rose, fellor remained unchanged. The levels were drawn pre-dose, 30 min, 60 min,120 min, 160 min and 180 min. The subject was sent home for a 3 day“washout” period and the same protocol was performed on the 100 mg/mlgel and then after a 3 day “washout” the same protocol was performed onthe 200 mg/ml gel.

Glutathione gel was applied to an area of approximately 60 squarecentimeters on the inside of the lower forearm. Prior to applyingglutathione gel the site was washed with soap and water and dried with atowel to eliminate any contaminating residue on the skin.

The first dose was applied at: 10:33 AM (50 mg)

The second dose was applied at: 11:25 AM (100 mg)

The third dose was applied at: 11:07 AM (200 mg)

Since the mechanical spray during testing did not always administer 100%accurate dosing the administrator manually measured the dose into asmall measurable plastic graduated cylinder. The amount of glutathioneproduct liquid to equal 50 mg for the first dose was precisely measured.The measurement was verified by the measurable applicator, which was apipette. The entire liquid drawn up in the pipette was applied to theforearm and rubbed in by the subject. This same process was used tomeasure out the 100 mg and the 200 mg dose. Blood levels/samplesobtained via blood draw in the anticutibal space of the arm. The onlychange observed from the subject's baseline assessment was fatigue thatlasted for approximately 7 hours the evening of the first dose. Itspontaneously resolved on its own.

Results:

The application of a single dose, (1 ml topical stabilized GSH gel) ofthe 200 mg/ml compound saw an increase in blood GSH of +392 μmol/L overa period of 180 minutes. The application of a single dose, (1 ml topicalstabilized GSH gel) of the 100 mg/ml compound saw an increase in bloodGSH of +102 μmol/L over a period of 180 minutes. The application of asingle dose, (1 ml topical stabilized GSH gel) of the 50 mg/ml compoundsaw a decrease in blood GSH of −70 μmol/L over a period of 180 minutes.This decrease could be caused by innumerable variables or by countlessphysiological processes and will not be discussed here. (FIG. 6A/B).

Blood level readings were also taken for superoxide dismutase (FIG.7A/B), glutathione peroxidase (FIG. 8A/B), and lipid peroxidase (FIG.9A/B) at 30, 60, 90, 120 and 180 minutes following doses of 50 mg/ml,100 mg/ml, and 200 mg/ml.

The study indicated that usable reduced glutathione entered the bloodtransdermally, impacting blood glutathione levels as well as levels ofrelated molecules. Therefore, this compound appears to cross the dermalbarrier and may be pharmaceutically useful as well as useful insignificantly lower doses including in cosmetics, antioxidant creams andnumerous other nutraceutical formulations.

Example 10: 34 Volunteer Nanonized GSH Double Blinded DetoxStudy—Synopsis

It was proposed that nanonized topical stable highly reduced glutathionewould act as a detoxification agent potentially removing heavy metals.It is unknown as to what percentage of the population suffers from heavymetal toxicity. There is no known natural heavy metal detoxificant sodetoxification could potentially be of interest for patients of kidneyor liver disease with broader implications regarding variousneurological disorders. Topical stable nanonized GSH is a patentedpharmaceutical grade product (RealGSH™ produced in the USA by The GluteGroup, LLC of Utah) that involves a Japanese (BioKyowa™) natural reducedglutathione (GSH) encased in a nano (γ-cyclodextrin ring from Cavamax™in Germany) ring, the encasement of which involves a process patent(currently pending). This was then placed in a sterile hydrous solutionfor topical application (from which it is easily applied). This productcaused no known side effect and its components (GSH and ring) areconsidered GRAS certified by the FDA.

The study involved 34 volunteers who were brought in in three groups.The volunteers collected urine for 24 hours in order to obtain abaseline urinalysis for quantitative heavy metals involving lead,mercury, and arsenic—testing used was an Electrothermal (Flameless) AASand Mercury Hydride procedure through Mountain Star ClinicalLaboratories was utilized in this study. On Day #2 the volunteers weregiven nanonized topical stable highly reduced glutathione (RealGSH™) at100 mgm/ml and told to apply it liberally throughout the day. They wereinstructed to wash off between applications but to be liberal in theirapplications. The study was double blinded but then was un-blinded andthe volunteers given placebo were crossed back over and followed forthree days and then given the third 24 urinalysis on Day #3. (FIGS. 12and 13).

Results: Approximately 75% of patients showed an increase in heavy metaloutput on Day #2 (the day that the nanonized GSH was applied) (FIGS. 10and 11). 60+% of the placebo crossovers showed a dramatic decrease inurine output of heavy metals (many from toxic levels during Day #1 andplacebo Day #2) on Day #3 (when they applied the nanonized GSH) (FIGS.12 and 13). This formulation of nanonized glutathione has also beenshown to cause improvement in kidney function in a number of patientsanecdotally.

Conclusions: This formulation of nanonized glutathione appears to be afairly effective heavy metal detox agent.

TABLE 2 Results of Before and After Study Lead Lead Calc Mercury PersonBefore After Before After Before After 1 10 10 28 28 5 5 2 10 10 10 13 55 4 10 10 19 21 6 6 5 10 10 20 29 7 5 6 10 10 19 18 6 5 7 10 10 16 35 55 8 11 10 17 30 5 5 9 10 10 9 12 5 7 14 10 10 22 14 5 5 15 10 10 16 15 55 19 10 10 16 17 5 5 20 10 10 12 14 5 5 21 10 10 22 20 6 5 22 10 10 2736 8 5 23 10 10 26 27 6 5 24 10 10 23 17 9 7 25 10 12 12 7 5 5 26 10 1114 11 5 5 28 11 10 16 17 5 5 30 10 10 24 43 5 5 31 10 10 5 28 5 5 32 1012 11 13 5 5 33 10 12 16 19 5 5 34 10 10 15 28 5 5 Average of Treated:10.08 10.29 17.29 21.33 5.54 5.21

TABLE 3 Results of Before and After Study Mercury Calc Arsenic ArsenicCalc Person Before After Before After Before After 1 14 14 17 20 47 56 25 6 17 21 17 26 4 12 12 5 8 10 15 5 14 15 5 5 10 16 6 11 9 12 5 23 9 7 817 10 7 16 24 8 8 15 5 5 8 15 9 5 8 24 14 22 16 14 11 7 5 5 11 7 15 8 77 5 11 7 19 8 9 6 5 9 9 20 6 7 5 5 6 7 21 13 10 7 5 16 10 22 22 18 7 719 25 23 16 14 8 8 21 22 24 20 12 5 27 11 45 25 6 3 15 12 18 7 26 7 5 95 12 5 28 7 8 13 12 9 24 30 12 22 14 15 33 65 31 3 14 5 7 3 19 32 5 5 55 5 5 33 8 8 5 5 8 8 34 8 14 12 11 18 31 Average of Treated: 9.88 10.799.29 9.33 15.13 19.71

TABLE 4 Results of Placebo Study Lead Lead Calc After After 3 days 3days Person Before Placebo GSH Before Placebo GSH  3 11 11 14 12 13 1110 10 10 10 30 31 26 11 10 10 10 26 25 28 12 11 10 10 17 21 13 13 10 1010 13 12 13 16 10 10 10 17 24 7 17 10 10 10 15 10 11 18 10 10 10 15 9 1127 10 10 17 28 32 27 29 10 10 10 14 29 27 Average: 10.20 10.10 11.1018.70 20.60 17.40

TABLE 5 Results of Placebo Study Mercury Mercury Calc After After 3 days3 days Person Before Placebo GSH Before Placebo GSH  3 5 5 5 5 6 4 10 55 5 15 16 13 11 5 5 5 13 13 14 12 5 5 5 8 11 6 13 5 5 5 7 6 6 16 6 5 510 12 4 17 6 5 5 9 5 5 18 5 5 5 8 4 3 27 5 5 5 14 16 14 29 5 5 5 7 14 13Average: 5.20 5.00 5.0 9.60 10.30 8.20

TABLE 6 Results of Placebo Study Arsenic Arsenic Calc After After 3 days3 days Person Before Placebo GSH Before Placebo GSH  3 5 5 7 5 6 5 10 910 12 27 31 31 11 10 5 10 26 13 28 12 15 11 12 23 23 15 13 12 7 7 16 7 916 50 34 22 83 82 16 17 5 5 5 8 5 5 18 8 7 5 12 6 3 27 39 31 18 108 9849 29 22 14 10 31 40 27 Average: 17.50 12.90 10.80 33.90 31.10 18.80

TABLE 7 Study Questionaire Number answering: Question: Yes No Change inEnergy? 7 5 Change in well being? 6 6 Change in mental clarity? 6 6Change in sleep? 4 8 Side effects? 2 10 Other benefits? 5 7

Example 11: ESRD in a 30 Year Old Male Improved to Normal withApplication of Topical Stable Nanonized Reduced Glutathione—A CaseReport

End-stage kidney disease (ESRD) is a chronic disease involving “thecomplete, or almost complete failure of the kidneys to function. Themain function of the kidneys is to remove wastes and excess water fromthe body. This occurs when the kidneys are no longer able to function ata level needed for day-to-day life. It usually occurs when chronickidney disease has worsened to the point at which kidney function isless than 10% of normal. ESRD almost always follows chronic kidneydisease. A person may have gradual worsening of kidney function for10-20 years or more before progressing to ESRD. Patients who havereached this stage need dialysis or a kidney transplant.

Topical stable nanonized GSH is a patented pharmaceutical grade product(RealGSH™ produced in the USA by The Glute Group, LLC of Utah) thatinvolves a Japanese (BioKyowa™) natural reduced glutathione (GSH)encased in a nano (γ-cyclodextrin ring from Cavamax™ in Germany) ring,the encasement of which involves a process patent (currently pending).This is then placed in a sterile hydrous solution for topicalapplication (from which it is easily applied). This product causes noknown side effect and its components (GSH and ring) are considered GRAScertified by the FDA We believe from our previous work and otherresearch produced (NIH review articles, etc.) reduced GSH is veryaggressively sticky at a molecular level—attaching to and removing fromthe body (via the kidneys) just about anything that should not be there(including, we've found in other studies, heavy metals such as lead,mercury, and arsenic). GSH has also been determined to be reduced inCKD/ESRD so elevating levels would hopefully improve GFR. RealGSH™ hasbeen shown upon application to immediately elevate skin and tissuelevels of GSH.

This case involved a 30 year old male with idiopathic chronic pulmonaryhypertension who had numerous radiographic tests requiring contrastmedia—the contrast media was felt to be most of the cause of his declinein renal function. He had a presenting GFR of 20 and creatinine of 2.85.The patient was told he would need to start on dialysis by a wellqualified nephrologist at a local university hospital. Topical stablenanonized glutathione (aqueous or hydrous) gel was applied by thepatient on a daily basis (approximately 200 mg a day) and the patienthad serial GFRs and renal function testing performed. Afterapproximately 4 weeks of application the creatinine returned to 2.19 andthe GFR returned to a level of 34 (both WNL). This is improvement ispresumably from the heavy metal removal secondary to increasing hisserum glutathione levels but stoichiometrically seems to go beyond that.These results indicate that this treatment would be a simple andinexpensive way to improve renal function in patients with ESRD.

Example 12: Clinical Study of Nanoglutathione Gel 200 mg/cc; Dr. MichaelH. Jensen, M.D.

The purpose of this report was to document pain relief with the use oftopical Nanoglutathione. Over 200 patients were tested onNanoglutathione gel 200 mg/cc during the past two years. Most of thesewere inpatients in long term care facilities, which allowed forcontinuing and accurate observation of results. This report explains theobserved effects of the use of topical nanoglutathione gel on patients,particularly relative to pain. Nanoglutathione is pure glutathione thathas been stabilized by a cyclodextrin ring and is used only topically.In concept, the glutathione is absorbed through the skin by the aid ofthe cyclodextrin ring and it has been demonstrated that actualglutathione levels rise significantly with this topical product. Thisreport covers the most recent five month period and does not representthe entire patient base on which this product has been tested.

Pain scales used in are zero to ten, with zero being no pain and tenbeing maximal pain. Each pump “squirt” of Nanoglutathione represents 50mg of Nanoglutathione.

“Shoulder” as used herein means the superior trapezius muscle unlessotherwise indicated.

Patient #1: A 54-year-old male with symptoms of fibromyalgia documentedby multiple doctors and this is a chronic problem. He was first treatedwith topical glutathione after an exacerbation of his fibromyalgia whichis constant. The affected areas treated were neck, shoulders andrhomboids; when I refer to shoulders in each case I really mean thesuperior trapezius muscle. Pain scale before treatment was 8/10; painscale after treatment was 5/10. This patient treated himself twice a daywith three squirts of glutathione total. After three days his pain scalewent to 3/10, and after two weeks 0/10. Duration of pain relief withfirst treatment was 4 hours, after two weeks 12 hours, and after threeweeks 36 hours.

Patient #2: A 49-year-old male with myofascial pain of the neck andshoulders, and lumbar spine area status post fairly recent surgicalfusion. The pain scale for neck and shoulders was 3/10, pain of thelower back 5/10. With two squirts of glutathione in each area thispatient had no relief.

Patient #3: A 41-year-old female with well documented standardfibromyalgia. Pain scale was 6/10 and after treatment 0/10. Squirts ofglutathione were to the trapezius and shoulders, two pumps per site;neck one pump. Length of relief was 4 to 6 hours. This patient treatedseveral times a day. Results were not observed to be cumulative butpatient experienced relief with each time treatment.

Patient #4: A 52-year-old male with acute trapezius and lumbar pain.Pain scale was 3/10, after treatment 0/10. Three squirts of glutathionewere used. Time to relief was 5 minutes; length of relief was 8 hours.

Patient #5: A 23-year-old male who is a body builder with symptoms ofmyofascial pain of biceps and right olecranon tendinitis. Pain scalebefore treatment was 4/10 and after treatment 0/10. Squirts ofglutathione were two to the biceps and two to the shoulders. Time torelief was 15 minutes. This patient received complete relief Symptomsreturned following repeat of heavy body building routine, but eachtreatment gave complete relief of pain.

Patient #6: A 76-year-old female with longstanding pain of the right hipthat is debilitating consistent with osteoarthritis. Pain scale beforetreatment was 5/10 and this pain was continuous. Pain scale aftertreatment was 0/10. Squirts of glutathione per treatment were two. Timeuntil relief was 2 hours. Length of pain relief was 12 hours. This hasbeen additive in that patient needs to repeat treatments only everyseveral days.

Patient #7: A 52-year-old female with acute tennis elbow. Pain scalebefore treatment was 5/10. Pain scale after treatment was 2. Time torelief was 30 minutes. Length of pain relief was 3 hours.

Patient #8: A 52-year-old female status post ground level fall withwhiplash type injury with pain of the neck and right shoulder. Painscale before treatment was 7/10. Pain scale after treatment was 2/10.Squirts of glutathione were two total. Time to relief was 20 minutes.Length of pain relief was 5 hours. This patient used glutathione twice aday for two weeks and had complete relief of symptoms.

Patient #9: A 13-year-old female with lumbar pain with localizedsciatica. Pain scale was 9/10, after treatment 4/10. Squirts ofglutathione per treatment were four twice a day. Time to relief was 30minutes. Length of relief was 5 hours.

Patient #10: A 43-year-old female with connective tissue disorder of thelumbar disks, or DJD, up her back and shoulder, fibromyofasciitis.Lumbar pain scale was 9/10 before treatment, after treatment 2/10.Shoulder and upper back pain 7/10, after treatment 2/10. Time to reliefwas 5 minutes. Length of pain relief was 4 hours.

Patient #11: A 43-year-old female with chronic lumbar pain withassociated sciatica. She was status post L4-5 fusion. Pain scale beforetreatment 8/10 of the lumbar spine and 6/10 of the sciatica. Aftertreatment lumbar pain was 0/10, the sciatica remained 6/10. Time torelief 10 minutes and squirts of glutathione were four.

Patient #12: A 54-year-old male with a right below-the-knee amputationwith associated phantom pain for many years. Pain scale before treatmentwas 8/10, after treatment was 2/10. Squirts of glutathione per treatmentwere two, twice a day. Time to relief was 30 minutes. Length of painrelief was 8 hours.

Patient #13: A 77-year-old female with chronic lumbar pain. This patientnotes degenerative joint disease of the lumbar spine, bone-on-bone, withleft sciatic nerve pain. Lower back pain before treatment was 9/10, lefthip or sciatic pain 9/10. After treatment, lumbar spine 3/10 and sciaticpain 3/10. Time to relief 6 minutes. Length of pain relief 4 hours.

Patient #14: A 43-year-old female with chronic left shoulder (leftsuperior trapezius) pain. SI pain times 10 years. Pain scale of boththese areas before treatment was 8/10, after treatment was 1/10. Time torelief 6 minutes. Squirts of glutathione were four total. Length ofrelief was 5 hours. This patient applied this twice a day and after twoweeks she only applies glutathione to these areas once every five days.Her pain is 0/10.

Patient #15: A 56-year-old female with neck and shoulder myofasciitis,left knee pain and this patient also has wrinkles on her face for whichshe applies glutathione. Pain scale of the neck before treatment was6/10, knee 7/10. After treatment neck pain was 2/10, knee pain 2/10.Squirts of glutathione to each area were two to face, two to neck andtwo to knee. Relief of pain of the neck and knee occurred in 15 minutes.She noticed increased muscle tone and decrease of wrinkles on her facein two weeks which is continuing. Length of pain relief was 6 to 8 hoursin the neck and knee. After three weeks of twice a day treatment sheused glutathione once every other day.

Patient #16: An 83-year-old male seen as an inpatient for right temporalpostherpetic neuralgia. This patient appeared to be dying with a painscale of 10/10 and was unable to eat. Pain before application to theright temporal area was 10/10, after 15 minutes was 2/10; this was thefirst application. Physician personally applied glutathione to thispatient every Monday, Wednesday, and Friday. Patient had cumulativerelief of pain over a period of three weeks and after a three weekperiod of single applications to this area on Monday, Wednesday, andFriday needed treatment only once a week. Initially the patient's painwas so severe that even flicking the patient's hair triggered screaming.Physician had this patient also evaluated by neurosurgery who toldPhysician that there was nothing wrong with the patient followingcompletion of the treatment regime. This patient continues to useglutathione once a week.

Patient #17: An 18-year-old, Polynesian, college football player withsprained left ribs after bench pressing and reps 450 pounds. Pain scalebefore treatment was 8/10, pain scale after treatment was 0/10. Squirtsof glutathione were one squirt one time. Time to relief was 5 minutes.This patient had complete resolution of his pain. Of note, this injurydid not occur over a single weight lifting session but he had had it forabout two weeks and was still continued to bench press.

Patient #18: A 42-year-old female with bilateral SI pain and right hippain for 10 years. Pain scale before treatment was 7/10, after treatmentwas 1-2/10. Squirts of glutathione were four total. These were appliedby Physician. Time to relief was 10 minutes. Length of relief 60 hours.After two weeks of b.i.d. treatment she now only uses glutathione everyfive days.

Patient #19: A 64-year-old male with myofasciitis of the rhomboidslasting several weeks. Pain scale before treatment was 6/10, aftertreatment 2/10. Squirts of glutathione per treatment were six. Timeuntil relief was 25 minutes. Length of relief was 6 hours. Physicianpersonally applied glutathione once every Tuesday and Thursday. Theresults were additive though pain relief remained at a 2/10.

Patient #20: A 60-year-old female with severe chronic plantar fasciitiswith associated neuropathy; osteoarthritis of the hands with associatedneuropathy, and osteoarthritis of the knees. Pain scale with the plantarfasciitis before treatment was 8/10, after treatment 0/10. Pain scale ofosteoarthritis of the hands with neuropathy before treatment was 6/10,after treatment 0/10. Pain scale of osteoarthritis of the knees beforetreatment was 6/10, after treatment 2/10. Time to relief in each casewas 30 minutes. Squirts of glutathione were two per foot, one per handand one per knee. Length of relief at the start was 24 hours, after twoweeks she used gluitathione only three days. Of note, the neuropathy ofher hands and of her feet was almost completely resolved to the pointthat stated it was barely detectable. This patient continues to useglutathione.

Patient #21: A 31-year-old female with right knee pain consistent withosteoarthritis. This patient weighed 300 pounds. She had knee painroughly three times a year which lasted at least a month. Pain scalebefore treatment was 5/10, after treatment was 0/10. Time to pain reliefwas 30 minutes. Squirts of glutathione were four. Of interest, thispatient only required one treatment and had no return of the pain.Physician continues to work with this patient three days a week.

Patient #22: A 72-year-old female with rheumatoid arthritis of the handsand major joints. Pain scale before treatment was generally 4/10 andthen down to 1/10. Squirts of glutathione per treatment were five. Timeuntil relief was 30 minutes. Length of pain relief was 8 to 12 hours.This has had an additive effect and the patient was able to decrease useof glutathione with the improvement in her hands.

Patient #23: An 82-year-old male with rheumatoid arthritis of the handsand major joints. Pain scale before treatment was 6/10, after treatment1/10. Time until relief was 30 minutes. Squirts of glutathione pertreatment were four. Length of pain relief was 12 hours. This patientnow uses glutathione only once every several days.

Conclusions

Nanoglutathione treats many types of pain including myofascial pain. InPhysician's experience with tension headaches, relief was in the 90thpercentile. Fibromyalgia duration of relief was 2 to 4 hours with nocures but with remarkable improvement. Neurogenic pain, including twocases with one postherpetic neuralgia case. This was a very extreme casewith the first application giving 90% resolution of pain. This patienthad complete resolution of pain and continues to use glutathione onceweek. With plantar fasciitis Physician's experience has been 100%effectiveness. Osteoarthritis was difficult as these are deep tissueswith varying results. Rheumatoid arthritis, there appeared to be ageneralized improvement of 30-50%. Deep back pain consistent withdegenerative disk disease, appeared to have 30-50% resolution requiringcontinued application.

Additionally, Physician experienced with two cases of second degreeburns. Pain relief occurred within 5 minutes and healing time appearedto be cut in half.

Physician remarked that a significant percentage of his clinicalpopulation had dementia and he observed that in 10-15% of casesapplication of glutathione, four to six squirts, created a remarkableincrease in mental acuity and wakefulness in all of these patients.Short term memory was also improved.

Example 13: RealGSH and Autism

It is known that approximately 50% of children born with autism spectrumdisorder are unable to methylate. Thus they cannot produce glutathione.By age 2 1 h to 3 years they start to show signs of their autism whichis actually secondary to heavy metal build-up internally in neurologicaltissue, which causes them symptoms of autism. Intravenous reducedglutathione (the only form previously thought to work) is too difficultand invasive to allow proper therapy with kids with autism. In oneclient intervention an 18 year old male, with advanced autism, had beenplaced in an Intermediate Care Facility for the Mentally Retarded(ICFMR) due to his paucity of speech (one word answers at most),inability to interact with others, and self-stimulation behavior. Thisclient, after family approval, was given a trial of one dose a day oftopical stable complexed glutathione (RealGSH) at approximately 200 mg aday. The study had intermediate videotaping of the patient (again withfamily release and approval) to document any potential improvement. Eachweek the participant met various improvement milestones and after 3months of therapy (90 days) the participant was asking questionsregarding where his family was, going shopping and making purchases andchange, interacting normally with other members of the staff and otherclients at the facility and elsewhere, and generally acting normally.

Example 14: RealGSH and Tardive Dyskinesia/Akathesia

Tardive dyskinesia is a difficult-to-treat form of dyskinesia, adisorder resulting in involuntary, repetitive body movements. In thisform of dyskinesia, the involuntary movements are tardive, meaning theyhave a slow or belated onset. This neurological disorder frequentlyappears after long-term or high-dose use of antipsychotic drugs, or inchildren and infants as a side effect from usage of drugs forgastrointestinal disorders.

Tardive dyskinesia is characterized by repetitive, involuntary,purposeless movements. Some examples of these types of involuntarymovements include grimacing, tongue protrusion, lip smacking, puckeringand pursing of the lips, and rapid eye blinking. Rapid, involuntarymovements of the limbs, torso, and fingers may also occur. In somecases, an individual's legs can be so affected that walking becomesdifficult or impossible. Respiratory irregularity, such as grunting anddifficulty breathing, is another symptom associated with tardivedyskinesia, although studies have shown that the prevalence rate isrelatively low.

In one client intervention a 61 year old female, with severe tardivedyskinesia, had been placed in an long term care facility (nursing home)due to her severe tongue thrusting, inability to communicate, inabilityto ambulate, and mental confusion. This client, after family approval,was given a trial of one dose a day of topical stable complexedglutathione (RealGSH) at approximately 200 mg a day. The study hadintermediate videotaping of the patient (again with family release andapproval) to document any potential improvement. Each week theparticipant met various improvement milestones and after 3 months oftherapy (90 days) the participant was speaking and asking questionsregarding where her family was, normal standing and ambulation, goingshopping and making purchases and change, interacting normally withother members of the staff and other clients at the facility andelsewhere, and generally acting normally. There is no effective therapyfor tardive dyskinesia.

Example 15: Stabilization of Nano Glutathione

Stabilized Glutathione, antioxidant and Gamma Cyclo-dextrine complex wasadjusted to a pH of not less than pH 4.00 and not more than pH 7.8, ornot less than pH 5.00 and not more than pH 7.20 using Sodium hydroxideor hydrochloric acid. The solution was nanosized using Ultrasonic waveswith appropriate ultrasonic equipment at about 100 Watts for not lessthan 1 minute and not more than 10 minutes, or less than 3 minutes andnot more than 6 minutes. The resulting nanoparticles were not less than20 angstroms and not more than 200 nanometers in size.

What is claimed is:
 1. A method of treating an inflammatory condition,comprising: identifying a target site on a subject affected by acondition associated with inflammation, and topically applying to thesubject a topical nanonized composition; wherein the compositioncomprises an inclusion complex comprising: reduced glutathione, anadditional antioxidant, and gamma cyclodextrin; wherein the reducedglutathione is non-esterified, non-acetylated, and non-fatty acidattached; wherein the reduced glutathione is stabilized in a reactionmixture by bringing the reduced glutathione and the additionalantioxidant in contact with the gamma cyclodextrin solubilized in apolar solution, wherein the molar ratio of reduced glutathione to gammacyclodextrin is between about 4:1 and 13:1; wherein the additionalantioxidant comprises at least one of the following: alpha-lipoic acid,ascorbic acid, uric acid, beta-carotene, alpha-tocopherol,dimethylethanolamine, CoEnzyme Q10, vitamin E, carnosine, colloidalsilver, catalase, superoxide dismutase, and peroxidase; and whereinultrasonic waves nanonize the inclusion complex into nanoparticles;wherein the nanoparticles have a particle size range between about 2 andabout 200 nanometers; and wherein the topical application of thenanonized complex to the target site facilitates rapid absorption of thereduced glutathione into the bloodstream of the subject.
 2. The methodof claim 1, wherein the reaction mixture is capped under vacuum aftermixing at a pH range of 3.0 to 7.0.
 3. The method of claim 1, whereinthe molar ratio of reduced glutathione to gamma cyclodextrin is betweenabout 8:1 and 10:1.
 4. The method of claim 1, wherein about 20% of thecomposition consists of reduced glutathione and about 10% of thecomposition consists of gamma cyclodextrin.
 5. The method of claim 1,wherein the additional antioxidant is ascorbic acid at a concentrationof about 4% by mass.
 6. The method of claim 1, wherein the polarsolution comprises ascorbic acid.
 7. The method of claim 6, wherein thepolar solution further comprises benzalkonium chloride.
 8. The method ofclaim 1, wherein the composition is formulated as a liquid, cream,lotion, oil, emulsion, spray, aerosol, dissolving strip, bolus, orsuppository.
 9. The method of claim 1, wherein the topical applicationcomprises sublingual, transdermal, cutaneous, subcutaneous, mucosal,transmucosal, inhalation, intralesional, or buccal routes.
 10. Themethod of claim 1, wherein the composition is administered to thesubject twice daily.
 11. The method of claim 1, wherein a daily dosageof the reduced glutathione is about 0.001 to 100 mg per kg of bodyweight of the subject.
 12. The method of claim 1, wherein theinflammatory condition is arthritis.
 13. The method of claim 1, whereinthe inflammatory condition is shingles.